US20210299280A1

US20210299280A1 - Compositions and methods for transient gene therapy with enhanced stability

Info

Publication number: US20210299280A1
Application number: US17/225,823
Authority: US
Inventors: Michael Goldberg; Ellese CARMONA
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 2015-06-05
Filing date: 2021-04-08
Publication date: 2021-09-30
Also published as: US20180169146A1; EP4219532A3; CA2987066A1; AU2016271519B2; EP3303377B1; EP3303377A1; WO2016197121A1; US11000547B2; AU2016271519A1; HK1253674A1; EP4219532A2

Abstract

The present invention provides circularized RNA and methods of making and using same.

Description

RELATED APPLICATIONS

This application is a division of U.S. Non-provisional application Ser. No. 15/579,322, filed Dec. 4, 2017, which is a national stage entry of PCT application No. PCT/US2016/036045 filed on Jun. 6, 2016, which claims priority to, and the benefit of, U.S. Provisional Application No. 62/171,538, filed Jun. 5, 2015, and U.S. Provisional Application No. 62/303,116, filed Mar. 3, 2016, the contents of each which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

This instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named “DFCI-108N01US_ST25.txt” created on Dec. 5, 2017 is 15 KB in size.

FIELD OF THE INVENTION

The present invention relates generally to compositions of circularized RNA, method of producing, and using same.

BACKGROUND OF THE INVENTION

Circular RNA is useful in the design and production of stable forms of RNA. The circularization of an RNA molecule provides an advantage to the study of RNA structure and function, especially in the case of molecules that are prone to folding in an inactive conformation (Wang and Ruffner, 1998). Circular RNA can also be particularly interesting and useful for in vivo applications, especially in the research area of RNA-based control of gene expression and therapeutics, including protein replacement therapy and vaccination.
Prior to this invention, there were three main techniques for making circularized RNA in vitro: splint-mediated method, permuted intron-exon method, and RNA ligase-mediated method.
However, the existing methodologies are limited by quantities of circularized RNA that can be produced and by the size of RNA that can be circularized, thus limiting their therapeutic application.
It is therefore a primary object of the current invention to provide a general method for preparation of a desired RNA in circularized form that is not limited by quantity or size.

SUMMARY OF THE INVENTION

An aspect of the present invention is a nucleic acid comprising: (a) a 5′ complement-reverse complement (CRC) sequence; (b) a 5′ untranslated region (UTR) sequence; (c) an RNA sequence; (d) a 3′ UTR sequence; and (e) a 3′ CRC sequence. The 5′ CRC sequence is at least partially complementary to the 3′CRC sequence, e.g., complementary to the 3′ CRC sequence.
In embodiments, the RNA sequence may be capable of being translated into a polypeptide, may comprise a RNA that is a reverse complement of an endogenous RNA, e.g., an mRNA, a miRNA, a tRNA, an rRNA, or a lncRNA, or may be capable of binding to an RNA-binding protein (RBP).
In embodiments, the nucleic acid may further comprise at least one random nucleotide sequence comprising between 5 and 25 nucleotides, e.g., 10 to 50 nucleotides, (e.g., 10, 15, or 20 nucleotides).
In embodiments, a 5′ random nucleotide sequence may be located at the nucleic acid's 5′ end and/or the 3′ random nucleotide sequence is located at the nucleic acid's 3′ end; the 5′ random nucleotide sequence may be located upstream of the 5′ CRC sequence and/or the 3′ random nucleotide sequence is located downstream of the 3′ CRC sequence. The 5′ random nucleotide sequence and the 3′ random nucleotide sequence may be at least partially complementary. Alternately, the 5′ random nucleotide sequence and the 3′ random nucleotide sequence may be non-complementary.
In embodiments, the nucleic acid may further comprise at least one 5′ and/or 3′ polyA sequence comprising between 5 and 25 nucleotides, e.g., 10 to 50 nucleotides (e.g., 10, 20, or 30 nucleotides), and located towards the nucleic acid's 5′ end and/or towards the nucleic acid's 3′ end. The 5′ polyA sequence may be located 5′ to the 5′ CRC sequence and/or the 3′ polyA sequence is located 3′ to the 3′ CRC sequence.
In embodiments, the 5′ and/or the 3′ CRC sequence may comprise 10 to 50 nucleotides, e.g., 10, 20, 30, or 40 nucleotides. Preferably, the 5′ and/or the 3′ CRC sequences comprise 20 nucleotides. The 5′ CRC sequence may comprise tggctgcacgaattgcacaa (SEQ ID NO: 1) and the 3′ CRC sequence may comprise ttgtgcaattcgtgcagcca (SEQ ID NO: 2).
In embodiments, the 5′ UTR may be polyAx30, polyAx120, PPT19, PPT19x4, GAAAx7, or polyAx30-EMCV. In embodiments, the 3′ UTR may be HbB1-PolyAx10, HbB1, HbB1x2, or a motif from the Elastin 3′ UTR, e.g., a 3′ UTR comprising the Elastin 3′ UTR or a motif thereof, e.g., which is repeated twice or three times. Preferably, the 5′ UTR is PPT19 or repeats thereof and the 3′ UTR is derived from Elastin or a motif thereof and/or repeats thereof
In embodiments, the RNA sequence may comprise at least 30 nucleotides, e.g., at least 300 nucleotides (e.g., at least 500 nucleotides). The polypeptide may comprise a tumor-associated antigen, a chimeric antigen receptor, a bacterial or viral antigen, a transposase or a nuclease, a transcription factor, a hormone, an scFv, a Fab, a single-domain antibody (sdAb), or a therapeutic protein. The therapeutic protein may be preproinsulin, hypocretin, human growth hormone, leptin, oxytocin, vasopressin, factor VII, factor VIII, factor IX, erythropoietin, G-CSF, alpha-galactosidase A, iduronidase, N-acetylgalactosamine-4-sulfatase, FSH, DNase, tissue plasminogen activator, glucocerebrosidase, interferon, or IGF-1. The polypeptide may comprise an epitope for presentation by an antigen presenting cell. The polypeptide may lead to improved T-cell priming, as determined by increased production of IFN-γ, including by proliferating cells.
In embodiments, the 5′ UTR may comprise an internal ribosome entry site (IRES); preferably, an encephalomyocarditis virus (EMCV) IRES or a PPT19 IRES.
In embodiments, the nucleic acid may comprise a modified nucleotide, e.g., 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl substituted naphthyl groups, an O- and N-alkylated purines and pyrimidines, N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, aminophenol, 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, or alkylcarbonylalkylated nucleotides. Preferably, the modified base is 5-methylcytidine (5mC).
In embodiments, the nucleic acid's 5′ and 3′ termini are not ligated, such that the nucleic acid is non-circularized.
In embodiments, the nucleic acid's 5′ and 3′ termini are ligated such that the nucleic acid is circularized. Such a circularized nucleic acid has greater stability (in vitro or in vivo) relative to a non-circularized nucleic acid; such a circularized nucleic acid provides greater polypeptide translation (in vitro or in vivo) relative to a non-circularized nucleic acid.
Another aspect of the present invention is a cell comprising any above-described nucleic acid, e.g., a circularized nucleic acid. A cell comprising an above-described circularized nucleic acid may further comprise a non-circularized nucleic acid having any above-described feature. The cell may be in vitro.
Yet another aspect of the present invention is a non-human mammal comprising an above-described cell, e.g., comprising a circularized nucleic acid or comprising a circularized nucleic acid and a non-circularized nucleic acid.
An aspect of the present invention is a primer comprising (a) a first motif comprising between 5 and 25 random nucleotides; (b) a second motif comprising a complement-reverse complement (CRC) sequence; (c) a third motif comprising an untranslated region (UTR) sequence; and (d) a fourth motif comprising about 20 nucleotides of a RNA sequence. In embodiments, the fourth motif encodes the first 20 nucleotides of the RNA sequence; alternately, the fourth motif encodes the last 20 nucleotides of the RNA sequence. In embodiments, the primer's RNA sequence may be capable of being translated into a polypeptide, may comprise a RNA that is a reverse complement of an endogenous RNA, e.g., an mRNA, a miRNA, a tRNA, an rRNA, or a lncRNA, or may be capable of binding to an RNA-binding protein (RBP).
Another aspect of the present invention is a method for circularizing a nucleic acid comprising: (a) obtaining any above-described nucleic acid and in which the nucleic acid is non-circularized; and (b) ligating the 5′ terminus of the nucleic acid to its 3′ terminus, thereby producing a circularized nucleic acid. In embodiments, the method may further comprise converting the 5′ triphosphate of the nucleic acid into a 5′ monophosphate, e.g., by contacting the 5′ triphosphate with RNA 5′ pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). Alternately, converting the 5′ triphosphate of the nucleic acid into a 5′ monophosphate may occur by a two-step reaction comprising: (a) contacting the 5′ nucleotide of the non-circularized nucleic acid with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the 5′ nucleotide after step (a) with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate. In embodiments, the ligating may occur by contacting the 5′ terminus of the nucleic acid and the 3′ terminus of the nucleic acid with a ligase, e.g., T4 RNA ligase. The ligating may be repeated at least one additional time, e.g., at least two additional times and at least three additional times. In embodiments, non-circularized nucleic acid molecules may be digested with an RNase, e.g., RNase R, Exonuclease T, λ Exonuclease, Exonuclease I, Exonuclease VII, T7 Exonuclease, or XRN-1; preferably, the RNase is RNase R and/or XRN-1. Non-circularized nucleic acid molecules may be digested with an RNase after the initial ligation or after the ligation is repeated at least one additional time. In embodiments, the obtained nucleic acid is synthesized by in vitro transcription (IVT).
Yet another aspect of the present invention is a circularized nucleic acid produced by an above-described method.
An aspect of the present invention is a composition comprising any above-described circularized nucleic acid. The composition may further comprise a non-circularized nucleic acid having any above-described feature.
Any of the above-described aspects or embodiments can be combined with any other aspect or embodiment as described herein.
Other features and advantages of the invention will be apparent from and encompassed by the Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a gel showing that enzymatic circularization produces the highest levels of circularized product (slower-migrating upper band) using existing protocols. Circularized product was never observed from the permuted intron-exon method, so these samples were not included in this gel.

FIGS. 2A and 2B include a gel and a table showing that RNA 5′ Pyrophosphohydrolase (RppH) treatment yields the highest level of circularized product compared to other monophosphate-generating enzymes.

FIGS. 3A and 3B include gel showing that RppH is the most efficient monophosphate-generating enzyme.

FIGS. 4A and 4B include a gel and a table showing that performing multiple rounds of ligase reaction increases the yield of circularized product.

FIGS. 5A and 5B include gels showing that RNase R and XRN-1 exonucleases result in the highest purity of circularized product.

FIGS. 6A to 6C include predicted secondary structures, a gel, and a table showing that inclusion of complement-reverse complement (CRC) motifs enhances efficiency of circularization of RNA constructs 300 to 500 nucleotides (NT) in length.

FIGS. 7A to 7D include predicted secondary structures, a gel, a table, and a graph showing that RNA up to 1000 nucleotide (NT) in length can be circularized when CRC sequences are included in the RNA molecule.

FIG. 8 includes a table showing that inclusion of CRC sequences enhances efficiency of circularization of RNA constructs up to at least 3000 NT in length.

FIGS. 9A to 9C include predicted secondary structures and gels showing that circularization efficiency is dependent on the 5′ and 3′ end positions of RNA molecules.

FIGS. 10A to 10C include gels and a table showing that inclusion of CRC sequences results in rapidly produced and robust levels of circularized product.

FIGS. 11A to 11C include a schematic of an RNA molecule and its predicted secondary structure, gels, and a graph showing that circularized RNA constructs containing a longer CRC and shorter Random NT motif confer the highest levels of circularization efficiency.

FIG. 12 includes a graph showing that shorter CRC results in higher translation efficiency of circularized RNA.

FIG. 13 includes a graph showing that longer CRC results in higher IFN-β response in Hep3Bs transfected with circularized RNA.

FIG. 14 includes a graph showing that a 20-mer derived from the elastin 3′ UTR motif enhances translation efficiency of linear and circularized RNA.

FIG. 15 includes graphs showing that the PPT19 (5′ UTR) and Elastinx3 (3′ UTR) combination sustain protein expression of circularized RNA.

FIG. 16 includes a graph showing that a 20-mer elastin 3′ UTR motif, but not the Hepatitis C Virus-derived (HCV) IRES (5′ UTR) or HbB1 (3′ UTR), enhances translation efficiency of circularized RNA.

FIG. 17 includes graphs showing that circularized RNA composed of 50% 5-methyl cytidine (5mC)-modified nucleotides confers high levels of translation.

FIG. 18 includes graphs showing that a modified nucleotide panel reveals that 50% 5mC composition provides the highest level of translation efficiency to circularized RNA.

FIG. 19 includes graphs showing that encephalomyocarditis virus (EMCV) IRES confers the greatest translation efficiency to circularized RNA but not to capped/tailed linear RNA.

FIG. 20 includes graphs showing that spiking with a competitive cap analog confirms that translation of circularized RNA is cap-independent.

FIG. 21 includes graphs showing that, in a cell-free system, circularized RNA containing the EMCV IRES is translated more efficiently than canonical (capped/tailed) linear mRNA.

FIG. 22 includes gels showing that circularized RNA is more resistant to degradation than canonical linear RNA is.

FIG. 23 includes a graph showing that, in vitro, circularized RNA encoding the therapeutic protein preproinsulin is sustained longer than linear RNA is. This finding is observed with multiple 5′ UTRs.

FIG. 24 includes a graph showing that, in vitro, circularized RNA is more stable than linear RNA is.

FIG. 25 includes graphs showing that, unlike linear RNA, circularized RNA shows minimal loss up to at least 3 days in mouse liver following intravenous administration.

FIG. 26 includes graphs showing that, in vivo, protein expression derived from circularized RNA is sustained longer than from linear RNA.

FIG. 27 includes a graph showing that, in vivo, circularized RNA is resistant to serum nucleases.

FIG. 28 includes a flow chart for experiments determining the kinetics of peptide presentation following transfection of dendritic cells (DCs) with circularized and/or linear RNA.

FIG. 29 includes a graph showing that, unlike for linear RNA, peptide presentation continues to increase beyond 2 days when circularized RNA is transfected into DCs.

FIG. 30 includes a flow chart for experiments relating to the co-culture of OT.I CD8 T cells with RNA-transfected DCs.

FIG. 31 includes flow cytometry plots showing a flow cytometry gating strategy after co-culture of OT.I CD8 T cells with RNA-transfected DCs.

FIG. 32 includes flow cytometry plots showing that addition of circularized RNA to linear RNA improved T cell priming (function, as assessed by IFN-γ).

FIG. 33 includes flow cytometry plots showing that addition of circularized RNA to linear RNA improved T cell priming (function among proliferating cells, as assessed by Ki67/IFN-γ double-positive cells).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides circularized nucleic acids (e.g. RNA), compositions comprising circularized nucleic acids, methods of circularizing nucleic acids, and methods using circularized nucleic acids. The nucleic acids, compositions, and methods are based upon the observation that circularization is more dependent on the availability of the free ends of the RNA than the size of the RNA construct.
RNA-based therapy affords benefits of gene therapy while remaining transient. Because RNA may be used as a transient, cytoplasmic expression system, RNA-based therapies can be applied in quiescent and/or slowly proliferating cells (i.e. muscle cells and hepatocytes). However, the instability of RNA, due to exonuclease-mediated degradation, has limited its clinical translation. In particular, the majority of RNA is degraded by exonucleases acting at both ends or at one end of the molecule after deadenylation and decapping. The sub-optimal stability of linear RNA remains an unresolved issue hindering the feasibility of RNA-based therapies. The majority of efforts to stabilize RNA have focused on linear RNA and modification thereof
Linear RNA is prone to exonuclease degradation from the 5′ to 3′ end and the 3′ to 5′ end, whereas circularized RNA transcripts have increased serum stability at least in part because the exonuclease binding sites are no longer accessible to exonucleases. However, there are currently no effective methods for producing large-scale circularized RNA suitable for therapeutic purposes. Prior techniques have relatively low yield, poor reproducibility of the reaction, and been limited by the size and types of RNA sequences that can be made circular.
In contrast, the current method possesses several new and advantageous features overcoming prior disadvantages encountered with other methods of creating circularized RNA. Indeed, the present invention provides, at least, the following advantages: 1) an optimized method for generating circularized RNA in higher yields than previously obtained; 2) circularized RNA encoding therapeutic proteins; 3) circularized RNA having improved stability (in solution, in cells, and in vivo); 4) longer circularized RNA molecules than previously obtained; 5) use of circularized RNA for therapeutic gene transfer into cells; 6) use of circularized RNA for improved vaccination; and 7) use of circularized non-coding RNA for binding to endogenous target RNAs and/or RNA-binding proteins.
The object of the present invention was to develop a method for preparation of a wide variety of circularized RNA molecules. Consequently, the present invention identified motifs in the 5′ and 3′ untranslated regions of the transcript that enable and enhance cap-independent translation.

Circularized RNA

The present invention is based upon 5′ and 3′ motifs that allow highly efficient enzymatic circularization of RNA. Specifically, it was discovered that complement-reverse complement (CRC) sequence motifs together with random nucleotides at the 5′ and 3′ ends of a desired RNA facilitates enzymatic circularization of RNA.
Accordingly, the invention provides a nucleic acid (DNA or RNA) comprising having a 5′ complement-reverse complement (CRC) sequence; a 5′ untranslated region (UTR) sequence; a RNA sequence; a 3′ untranslated region (UTR) sequence; and a 3′ CRC sequence. The RNA sequence may be an RNA sequence capable of being translated into a polypeptide; the RNA sequence may be a non-coding RNA e.g., an RNA that is a reverse complement of an endogenous RNA, i.e., an mRNA, a miRNA, a tRNA, an rRNA, or a lncRNA; or the RNA sequence may be capable of binding to an RNA-binding protein (RBP). When the RNA sequence binds an RBP, the nucleic acid of the present invention prevents the RBP from binding to its canonical linear RNA binding partner. In a nucleic acid, a 5′ CRC sequence may be partially complementary to a 3′ CRC sequence (i.e., including at least one pair of complementary nucleotides but not necessarily completely complementary).
The 5′ or 3′ complement-reverse complement sequence comprises 10 to 50 nucleotides, e.g., 10, 20, 30, 40, and 50 nucleotides. Preferably, the CRC sequence comprises the nucleotide sequence: tggctgcacgaattgcacaa (SEQ ID NO: 1) or ttgtgcaattcgtgcagcca (SEQ ID NO: 2).
The nucleic acid further incudes a random nucleotide sequence at the 5′ end and the 3′ end. The 5′ random nucleotide sequence is upstream of the 5′ CRC sequence, and the 3′ random nucleotide sequence is downstream of the 3′ CRC sequence.
Each random nucleotide sequence is between about 5 and 50 nucleotides, e.g., 10, 15, 20, and 25 nucleotides.
Rather than having random nucleotide sequences, a nucleic acid may have one or two polyA sequences, with the polyA sequences being upstream of a 5′ CRC and/or downstream of a 3′ CRC and at the nucleic acid's end(s).
Each polyA sequence is between about 5 and 50 nucleotides, e.g., 10, 15, 20, 25, and 30 nucleotides.
Preferred 5′ or 3′ complement-reverse complement sequences and random nucleotide sequences are exemplified in TABLE 1.

TABLE 1

	Primer Name		Primer				First 10 NT of	Last 10 NT of				Reverser	Primer Name
#	(Forward)	Vector	Size	T7	5′ Random	5′ CRC	PPT19x4	ElastinX3	3′ CRC	3′ Random	Size	Complement	(Reverse)

1	558_Hete	Hetero	47	Taatacga	GGGAATC	TGCCGT	AAAAGAA	Gaggga	ACCGA	CGGAAT	30	CTATATTCC	559 Het
	ro_10-			ctcactata	GAC (SEQ	CGGT	GGA (SEQ	ggga	CGGCA	ATAG		GTGCCGTCG	ero_10-
	10_F			(SEQ ID	ID NO: 30)	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	(SEQ ID		GTtccctccctc	10_R
				NO: 9)		NO 18)		NO: 7)	NO: 17)	NO: 24)		(SEQ ID NO:
												39)

2	560_Hete	Hetero	52	Taatacga	GGGAATC	TGCCGT	AAAAGAA	Gaggga	ACCGA	CGGAAT	35	TGCTTCTATA	561_Het
	ro_15-			ctcactata	GACTACA	CGGT	GGA (SEQ	ggga	CGGCA	ATAG		TTCCGTGCC	ero 15-
	10_F			(SEQ ID	G (SEQ ID	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	(SEQ ID		GTCGGTtccc	10_R
				NO: 9)	NO: 31)	NO: 18)		NO: 7)	NO: 17)	AAGCA		tccctc (SEQ
										NO: 25)		ID NO: 51)

3	562_Hete	Hetero	57	Taatacga	GGGAATC	TGCCGT	AAAAGAA	Gaggga	ACCGA	CGGAAT	40	TCTTATGCTT	563 Het
	ro_20-			ctcactata	GACTACA	CGGT	GGA (SEQ	ggga	CGGCA	ATAG		CTATATTCC	ero_20-
	10_F			(SEQ ID	GGAGGA	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	AAGCAT		GTGCCGTCG	10_R
				NO: 9)	(SEQ ID	NO: 18)		NO: 7)	NO: 17)	AAGA		GTtccctccctc
					NO: 32)					(SEQ ID		(SEQ ID NO:
										NO: 26)		45)

4	564_Hete	Hetero	47	Taatacga	GGGAAAA	TGCCGT	AAAAGAA	Gaggga	ACCGA	AAAAAA	30	TTTTTTTTTT	565 Het
	ro_10A-			ctcactata	AAA (SEQ	CGGT	GGA (SEQ	ggga	CGGCA	AAAA		TGCCGTCGG	ero_10A
	10_F			(SEQ ID	ID NO: 27)	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	(SEQ ID		Ttccctccctc	-10_R
				NO: 9)		NO: 18)		NO: 7)	NO: 17)	NO: 14)		(SEQ ID NO:
												57)

5	566_Hete	Hetero	52	Taatacga	GGGAAAA	TGCCGT	AAAAGAA	Gaggga	ACCGA	AAAAAA	35	TTTTTTTTTT	567 Het
	ro_15A-			ctcactata	AAAAAAA	CGGT	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTTGCCG	ero_15A
	10_F			(SEQ ID	A (SEQ ID	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	AAA (SEQ		TCGGTtccctc	-10_R
				NO: 9)	NO: 28)	NO: 18)		NO: 7)	NO: 17)	ID NO:		cctc (SEQ ID
										15)		NO: 63)

6	568_Hete	Hetero	57	Taatacga	GGGAAAA	TGCCGT	AAAAGAA	Gaggga	ACCGA	AAAAAA	40	TTTTTTTTTT	569 Het
	ro_20A-			ctcactata	AAAAAAA	CGGT	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTTTTTT	ero_20A
	10_F			(SEQ ID	AAAAAA	(SEQ ID	ID NO: 6)	(SEQ ID	(SEQ ID	AAAAAA		TGCCGTCGG	-10_R
				NO: 9)	(SEQ ID	NO: 18)		NO: 7)	NO: 17)	AA (SEQ		Ttccctccctc
					NO: 29)					ID NO:		(SEQ ID NO:
										16)		69)

7	570_Hete	Hetero	57	Taatacga	GGGAATC	AACACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	40	CTATATTCC	571 Het
	ro_10-			ctcactata	GAC (SEQ	TTAT	GGA (SEQ	ggga	CGGCA	ATAG		GAACACGTT	era 10-
	20_F			(SEQ ID	ID NO: 30)	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		ATTGCCGTC	20_R
				NO: 9)		CGGT		NO: 7)	GTGTT	NO: 24)		GGTtccctccct
						(SEQ ID			(SEQ ID			c (SEQ ID
						NO: 19)			NO: 20)			NO: 34)

8	572_Hete	Hetero	62	Taatacga	GGGAATC	AACACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	45	TGCTTCTATA	573_Het
	ro_15-			ctcactata	GACTACA	TTAT	GGA (SEQ	ggga	CGGCA	ATAG		TTCCGAACA	ero_15-
	20_F			(SEQ ID	G (SEQ ID	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	AAGCA		CGTTATTGC	20_R
				NO: 9)	NO: 31)	CGGT		NO: 7)	GTGTT	(SEQ ID		CGTCGGTtcc
						(SEQ ID			(SEQ ID	NO: 25)		ctccctc (SEQ
						NO: 19)			NO: 20)			ID NO: 46)

9	574_Hete	Hetero	67	Taatacga	GGGAATC	AACACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	50	TCTTATGCTT	575_Het
	ro_20-			ctcactata	GACTACA	TTAT	GGA (SEQ	ggga	CGGCA	ATAG		CTATATTCC	ero_20-
	20_F			(SEQ ID	GGAGGA	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	AAGCAT		GAACACGTT	20_R
				NO: 9)	(SEQ ID	CGGT		NO: 7)	GTGTT	AAGA		ATTGCCGTC
					NO: 32)	(SEQ ID			(SEQ ID	(SEQ ID		GGTtccctccct
						NO: 19)			NO: 20)	NO: 26)		c (SEQ ID
												NO: 40)

10	576_Hete	Hetero	57	Taatacga	GGGAAAA	AACACG	AAAAGAA	Gaggga	ACCGA	GGGAAA	40	TTTTTTTCCC	577_Het
	ro_10A-			ctcactata	AAA (SEQ	TTAT	GGA (SEQ	ggga	CGGCA	AAAA		AACACGTTA	ero_10A
	20_F			(SEQ ID	ID NO: 27)	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		TTGCCGTCG	-20_R
				NO: 9)		CGGT		NO: 7)	GTGTT	NO: 27)		GTtccctccctc
						(SEQ ID			(SEQ ID			(SEQ ID NO:
						NO: 19)			NO: 20)			52)

11	578_Hete	Hetero	62	Taatacga	GGGAAAA	AACACG	AAAAGAA	Gaggga	ACCGA	GGGAAA	45	TTTTTTTTTT	579_Het
	ro_15A-			ctcactata	AAAAAAA	TTAT	GGA (SEQ	ggga	CGGCA	AAAAAA		TTCCCAACA	ero_15A
	20_F			(SEQ ID	A (SEQ ID	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	AAA (SEQ		CGTTATTGC	-20_R
				NO: 9)	NO: 28)	CGGT		NO: 7)	GTGTT	ID NO:		CGTCGGTtcc
						(SEQ ID			(SEQ ID	28)		ctccctc (SEQ
						NO: 19)			NO: 20)			ID NO: 58)

12	580_Hete	Hetero	67	Taatacga	GGGAAAA	AACACG	AAAAGAA	Gaggga	ACCGA	GGGAAA	50	TTTTTTTTTT	581_Het
	ro_20A-			ctcactata	AAAAAAA	TTAT	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTTTCCC	ero_20A
	20_F			(SEQ ID	AAAAAA	TGCCGT	ID NO: 6)	(SEQ ID	ATAAC	AAAAAA		AACACGTTA	-20_R
				NO: 9)	(SEQ ID	CGGT		NO: 7)	GTGTT	AA (SEQ		TTGCCGTCG
					NO: 29)	(SEQ ID			(SEQ ID	ID NO:		GTtccctccctc
						NO: 19)			NO: 20)	29)		(SEQ ID NO:
												64)

13	582_Hete	Hetero	67	Taatacga	GGGAATC	GTTACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	50	CTATATTCC	583_Het
	ro_10-			ctcactata	GAC (SEQ	TACC	GGA (SEQ	ggga	CGGCA	ATAG		GGTTACGTA	ero_10-
	30_F			(SEQ ID	ID NO: 30)	AACACG	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		CCAACACGT	30_R
				NO: 9)		TTAT		NO: 7)	GTGTT	NO: 24)		TATTGCCGT
						TGCCGT			GGTAC			CGGTtccctcc
						CGGT			GTAAC			ctc (SEQ ID
						(SEQ ID			(SEQ ID			NO: 38)
						NO: 33)			NO: 21)

14	584_Hete	Hetero	72	Taatacga	GGGAATC	GTTACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	55	TGCTTCTATA	585_Het
	ro_15-			ctcactata	GACTACA	TACC	GGA (SEQ	ggga	CGGCA	ATAG		TTCCGGTTA	ero_15-
	30_F			(SEQ ID	G (SEQ ID	AACACG	ID NO: 6)	(SEQ ID	ATAAC	AAGCA		CGTACCAAC	30_R
				NO: 9)	NO: 31)	TTAT		NO: 7)	GTGTT	(SEQ ID		ACGTTATTG
						TGCCGT			GGTAC	NO: 25)		CCGTCGGTtc
						CGGT			GTAAC			cctccctc
						(SEQ ID			(SEQ ID			(SEQ ID NO:
						NO: 33)			NO: 21)			50)

15	586_Hete	Hetero	77	Taatacga	GGGAATC	GTTACG	AAAAGAA	Gaggga	ACCGA	CGGAAT	60	TCTTATGCTT	587_Het
	ro_20-			ctcactata	GACTACA	TACC	GGA (SEQ	ggga	CGGCA	ATAG		CTATATTCC	ero_20-
	30_F			(SEQ ID	GGAGGA	AACACG	ID NO: 6)	(SEQ ID	ATAAC	AAGCAT		GGTTACGTA	30_R
				NO: 9)	(SEQ ID	TTAT		NO: 7)	GTGTT	AAGA		CCAACACGT
					NO: 32)	TGCCGT			GGTAC	(SEQ ID		TATTGCCGT
						CGGT			GTAAC	NO: 26)		CGGTtccctcc
						(SEQ ID			(SEQ ID			ctc (SEQ ID
						NO: 33)			NO: 21)			NO: 44)

16	588_Hete	Hetero	67	Taatacga	GGGAAAA	GTTACG	AAAAGAA	Gaggga	ACCGA	AAAAAA	50	TTTTTTTTTT	589_Het
	ro_10A-			ctcactata	AAA (SEQ	TACC	GGA (SEQ	ggga	CGGCA	AAAA		GTTACGTAC	ero_10A
	30_F			(SEQ ID	ID NO: 27)	AACACG	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		CAACACGTT	-30_R
				NO: 9)		TTAT		NO: 7)	GTGTT	NO: 14)		ATTGCCGTC
						TGCCGT			GGTAC			GGTtccctccct
						CGGT			GTAAC			c (SEQ ID
						(SEQ ID			(SEQ ID			NO: 56)
						NO: 33)			NO: 21)

17	590_Hete	Hetero	72	Taatacga	GGGAAAA	GTTACG	AAAAGAA	Gaggga	ACCGA	AAAAAA	55	TTTTTTTTTT	591_Het
	ro-15A-			ctcactata	AAAAAAA	TACC	GGA (SFO	ggga	CGGCA	AAAAAA		TTTTTGTTAC	ero_15A
	30_F			(SEQ ID	A (SEQ ID	AACACG	ID NO: 6)	(SEQ ID	ATAAC	AAA (SEQ		GTACCAACA	-30_R
				NO: 9)	NO: 28)	TTAT		NO: 7)	GTGTT	ID NO:		CGTTATTGC
						TGCCGT			GGTAC	15)		CGTCGGTtcc
						CGGT			GTAAC			ctccctc (SEQ
						(SEQ ID			(SEQ ID			ID NO: 62)
						NO: 33)			NO: 21)

18	592_Hete	Hetero	77	Taatacga	GGGAAAA	GTTACG	AAAAGAA	Gaggga	ACCGA	AAAAAA	60	TTTTTTTTTT	593_Het
	ro_20A-			ctcactata	AAAAAAA	TACC	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTTTTTT	ero_20A
	30_F			(SEQ ID	AAAAAA	AACACG	ID NO: 6)	(SEQ ID	ATAAC	AAAAAA		GTTACGTAC	-30_R
				NO: 9	(SEQ ID	TTAT		NO: 7)	GTGTT	AA (SEQ		CAACACGTT
					NO: 29)	TGCCGT			GGTAC	ID NO:		ATTGCCGTC
						CGGT			GTAAC	16)		GGTtccctccct
						(SEQ ID			(SEQ ID			c (SEQ ID
						NO: 33)			NO: 21)			NO: 68)

19	594_Hete	Hetero	77	Taatacga	GGGAATC	AGGTTC	AAAAGAA	Gaggga	ACCGA	CGGAAT	60	CTATATTCC	595_Het
	ro-10-			ctcactata	GAC (SEQ	GAAG	GGA (SEQ	ggga	CGGCA	ATAG		GAGGTTCGA	ero_10-
	40_F			(SEQ ID	ID NO: 30)	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		AGGTTACGT	40_R
				NO: 9)		TACC		NO: 7)	GTGTT	NO: 24)		ACCAACACG
						AACACG			GGTAC			TTATTGCCG
						TTAT			GTAAC			TCGGTtccctc
						TGCCGT			CTTCG			cctc (SEQ ID
						CGGT			AACCT			NO: 35)
						(SEQ ID			(SEQ ID
						NO: 23)			NO: 22)

20	596_Hete	Hetero	82	Taatacga	GGGAATC	AGGTTC	AAAAGAA	Gaggga	ACCGA	CGGAAT	65	TGCTTCTATA	597_Het
	ro_15-			ctcactata	GACTACA	GAAG	GGA (SEQ	ggga	CGGCA	ATAG		TTCCGAGGT	ero_15-
	40_F			(SEQ ID	G (SEQ ID	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	AAGCA		TCGAAGGTT	40_R
				NO: 9)	NO: 31)	TACC		NO: 7)	GTGTT	(SEQ ID		ACGTACCAA
						AACACG			GGTAC	NO: 25)		CACGTTATT
						TTAT			GTAAC			GCCGTCGGT
						TGCCGT			CTTCG			tccctccctc
						CGGT			AACCT			(SEQ ID NO:
						(SEQ ID			(SEQ ID			47)
						NO: 23)			NO: 22)

21	598_Hete	Hetero	87	Taatacga	GACTACA	AGGTTC	AAAAGAA	Gaggga	ACCGA	CGGAAT	70	TCTTATGCTT	599_Het
	ro_20-			ctcactata	GGAGGA	GAAG	GGA (SEQ	ggga	CGGCA	ATAG		CTATATTCC	ero_20-
	40_F			(SEQ ID	(SEQ ID	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	AAGCAT		GAGGTTCGA	40_R
				NO: 9)	NO: 32)	GGGAATC		NO: 7)	GTGTT	AAGA		AGGTTACGT
						TACC			GGTAC	(SEQ ID		ACCAACACG
						AACACG			GTAAC	NO: 26)		TTATTGCCG
						TTAT			CTTCG			TCGGTtccctc
						TGCCGT			AACCT			cctc (SEQ ID
						CGGT			(SEQ ID			NO: 41)
						(SEQ ID			NO: 22)
						NO: 23)

22	600_Hete	Hetero	77	Taatacga	GGGAAAA	AGGTTC	AAAAGAA	Gaggga	ACCGA	AAAAAA	60	TTTTTTTTTT	601_Het
	ro_10A-			ctcactata	AAA (SEQ	GAAG	GGA (SEQ	ggga	CGGCA	AAAA		AGGTTCGAA	ero_10A
	40_F			(SEQ ID	ID NO: 27)	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	(SEQ ID		GGTTACGTA	-40_R
				NO: 9)		TACC		NO: 7)	GTGTT	NO: 14)		CCAACACGT
						AACACG			GGTAC			TATTGCCGT
						TTAT			GTAAC			CGGTtccctcc
						TGCCGT			CTTCG			ctc (SEQ ID
						CGGT			AACCT			NO: 53)
						(SEQ ID			(SEQ ID
						NO: 23)			NO: 22)

23	602_Hete	Hetero	82	Taatacga	GGGAAAA	AGGTTC	AAAAGAA	Gaggga	ACCGA	AAAAAA	65	TTTTTTTTTT	603_Het
	ro_15A-			ctcactata	AAAAAAA	GAAG	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTAGGTT	ero_15A
	40_F			(SEQ ID	A (SEQ ID	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	AAA (SEQ		CGAAGGTTA	-40_R
				NO: 9)	NO: 28)	TACC		NO: 7)	GTGTT	ID NO:		CGTACCAAC
						AACACG			GGTAC	15)		ACGTTATTG
						TTAT			GTAAC			CCGTCGGTtc
						TGCCGT			CTTCG			cctccctc
						CGGT			AACCT			(SEQ ID NO:
						(SEQ ID			(SEQ ID			59)
						NO: 23)			NO: 22)

24	604_Hete	Hetero	87	Taatacga	GGGAAAA	AGGTTC	AAAAGAA	Gaggga	ACCGA	AAAAAA	70	TTTTTTTTTT	605_Het
	ro_20A-			ctcactata	AAAAAAA	GAAG	GGA (SEQ	ggga	CGGCA	AAAAAA		TTTTTTTTTT	ero_20A
	40_F			(SEQ ID	AAAAAA	GTTACG	ID NO: 6)	(SEQ ID	ATAAC	AAAAAA		AGGTTCGAA	-40_R
				NO: 9)	(SEQ ID	TACC		NO: 7)	GTGTT	AA (SEQ		GGTTACGTA
					NO: 29)	AACACG			GGTAC	ID NO:		CCAACACGT
						TTAT			GTAAC	16)		TATTGCCGT
						TGCCGT			CTTCG			CGGTtccctcc
						CGGT			AACCT			ctc (SEQ ID
						(SEQ ID			(SEQ ID			NO: 65)
						NO: 23)			NO: 22)

25	606_Hom	Homo	47	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	30	CTATATTCC	607_Ho
	o_10-			ctcactata	GAC (SEQ	cc (SEQ	GGA (SEQ	ggga	gggg	ATAG		Gcccccccccct	mo_10-
	10_F			(SEQ ID	ID NO: 30)	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	(SEQ ID		ccctccctc	10_R
				NO: 9)		10)		NO: 7)	NO: 12)	NO: 24)		(SEQ ID NO:
												36)

26	608_Hom	Homo	52	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	35	TGCTTCTATA	609_Ho
	o_15-			ctcactata	GACTACA	cc (SEQ	GGA (SEQ	ggga	gggg	ATAG		TTCCGcccccc	mo_15-
	10_F			(SEQ ID	G (SEQ ID	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	AAGCA		cccctccctccct	10_R
				NO: 9)	NO: 31)	10)		NO: 7)	NO: 12)	(SEQ ID		c (SEQ ID
										NO: 25)		NO: 49)

27	610_Hom	Homo	57	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	40	TCTTATGCTT	611_Ho
	o_20			ctcactata	GACTACA	cc (SEQ	GGA (SEQ	ggga	gggg	ATAG		CTATATTCC	mo _20-
	10_F			(SEQ ID	GGAGGA	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	AAGCAT		Gcccccccccct	10_R
				NO: 9)	(SEQ ID	10)		NO: 7)	NO: 12)	AAGA		ccctccctc
					NO: 32)					(SEQ ID		(SEQ ID NO:
										NO: 26)		43)

28	612_Hom	Homo	47	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	30	TTTTTTTTTTc	613_Ho
	o_10A-			ctcactata	AAA (SEQ	cc (SEQ	GGA (SEQ	ggga	gggg	AAAA		ccccccccctcc	mo_10A
	10_F			(SEQ ID	ID NO: 27)	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	(SEQ ID		ctccctc (SEQ	-10_R
				NO: 9)		10)		NO: 7)	NO: 12)	NO: 14)		ID NO: 55)

29	614_Hom	Homo	52	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	35	TTTTTTTTTT	615_Ho
	o_15A-			ctcactata	AAAAAAA	cc (SEQ	GGA (SEQ	ggga	gggg	AAAAAA		TTTTTcccccc	mo_15A
	10_F			(SEQ ID	A (SEQ ID	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	AAA (SEQ		cccctccctccct	-10_R
				NO: 9)	NO: 28)	10)		NO: 7)	NO: 12)	ID NO:		c (SEQ ID
										15)		NO: 61)

30	616_Hom	Homo	57	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	40	TTTTTTTTTT	617_Ho
	o 20A-			ctcactata	AAAAAAA	cc (SEQ	GGA (SEQ	ggga	gggg	AAAAAA		TTTTTTTTTTc	mo_20A
	10_F			(SEQ ID	AAAAAA	ID NO:	ID NO: 6)	(SEQ ID	(SEQ ID	AAAAAA		ccccccccctcc	-10_R
				NO: 9)	(SEQ ID	10)		NO: 7)	NO: 12)	AA (SEQ		ctccctc (SEQ
					NO: 29)					ID NO:		ID NO: 67)
										16)

31	618_Hom	Homo	77	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	60	CTATATTCC	619_Ho
	o_10-			ctcactata	GAC (SEQ	cccccccc	GGA (SEQ	ggga	gggggg	ATAG		Gccccccccccc	mo_10-
	40_F			(SEQ ID	ID NO: 30)	cccccccc	ID NO: 6)	(SEQ ID	gggggg	(SEQ ID		cccccccccccc	40_R
				NO: 9)		cccccccc		NO: 7)	gggggg	NO: 24)		cccccccccccc
						cccccccc			gggggg			ccccctccctcc
						(SEQ ID			gggggg			ctc (SEQ ID
						NO: 11)			gggg			NO: 37)
									(SEQ ID
									NO: 13)

32	620_Hom	Homo	82	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	65	TGCTTCTATA	621_Ho
	o_15-			ctcactata	GACTACA	cccccccc	GGA (SEQ	ggga	gggggg	ATAG		TTCCGcccccc	mo_15-
	40_F			(SEQ ID	G (SEQ ID	cccccccc	ID NO: 6)	(SEQ ID	gggggg	AAGCA		cccccccccccc	40_R
				NO: 9)	NO: 31)	cccccccc		NO: 7)	gggggg	(SEQ ID		cccccccccccc
						cccccccc			gggggg	NO: 25)		cccccccccctc
						(SEQ ID			gggggg			cctccctc
						NO: 11)			gggg			(SEQ ID NO:
									(SEQ ID			48)
									NO: 13)

33	622_Hom	Homo	87	Taatacga	GGGAATC	Cccccccc	AAAAGAA	Gaggga	Gggggg	CGGAAT	70	TCTTATGCTT	623_Ho
	o_20-			ctcactata	GACTACA	cccccccc	GGA (SEQ	ggga	gggggg	ATAG		CTATATTCC	mo_20-
	40_F			(SEQ ID	GGAGGA	cccccccc	ID NO: 6)	(SEQ ID	gggggg	AAGCAT		Gccccccccccc	40_R
				NO: 9)	(SEQ ID	cccccccc		NO: 7)	gggggg	AAGA		cccccccccccc
					NO: 32)	cccccccc			gggggg	(SEQ ID		cccccccccccc
						(SEQ ID			gggggg	NO: 26)		ccccctccctcc
						NO: 11)			gggg			ctc (SEQ ID
									(SEQ ID			NO: 42)
									NO: 13)

34	624_Hom	Homo	77	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	60	TTTTTTTTTTc	625_Ho
	o_10A-			ctcactata	AAA (SEQ	cccccccc	GGA (SEQ	ggga	gggggg	AAAA		cccccccccccc	mo_10A
	40_F			(SEQ ID	ID NO: 27)	cccccccc	ID NO: 6)	(SEQ ID	gggggg	(SEQ ID		cccccccccccc	-40_R
				NO: 9)		cccccccc		NO: 7)	gggggg	NO: 14)		cccccccccccc
						cccccccc			gggggg			ccctccctccctc
						(SEQ ID			gggggg			(SEQ ID NO:
						NO: 11)			gggg			54)
									(SEQ ID
									NO: 13)

35	626 _Hom	Homo	82	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	65	TTTTTTTTTT	627_Ho
	o_15A-			ctcactata	AAAAAAA	cccccccc	GGA (SEQ	ggga	gggggg	AAAAAA		TTTTTcccccc	mo_15A
	40_F			(SEQ ID	A (SEQ ID	cccccccc	ID NO: 6)	(SEQ ID	gggggg	AAA (SEQ		cccccccccccc	-40_R
				NO: 9)	NO: 28)	cccccccc		NO: 7)	gggggg	ID NO:		cccccccccccc
						cccccccc			gggggg	15)		cccccccccctc
						(SEQ ID			gggggg			cctccctc
						NO: 11)			gggg			(SEQ ID NO:
									(SEQ ID			60)
									NO: 13)

36	628_Hom	Homo	87	Taatacga	GGGAAAA	Cccccccc	AAAAGAA	Gaggga	Gggggg	AAAAAA	70	TTTTTTTTTT	629_Ho
	o_20A-			ctcactata	AAAAAAA	cccccccc	GGA (SEQ	ggga	gggggg	AAAAAA		TTTTTTTTTTc	mo_20A
	40_F			(SEQ ID	AAAAAA	cccccccc	ID NO: 6)	(SEQ ID	gggggg	AAAAAA		cccccccccccc	-40_R
				NO: 9)	(SEQ ID	cccccccc		NO: 7)	gggggg	AA (SEQ		cccccccccccc
					NO: 29)	cccccccc			gggggg	ID NO:		cccccccccccc
						(SEQ ID			gggggg	16)		ccctccctccctc
						NO: 11)			gggg			(SEQ ID NO:
									(SEQ ID			66)
									NO: 13)

37	630_OG_	Origin-	60	Taatacga	Ttatgataa	Tggctgca	AAAAGAA	Gaggga	Ttgtgca	Agcgacttc	40	Cgaagtcgcttg	631_OG
	10-20_F	al		ctcactata	c (SEQ ID	cgaattgc	GGA (SEQ	ggga	attcgtg	g (SEQ ID		gctgcacgaatt	_10-
				ggg (SEQ	NO: 4)	acaa	ID NO: 6)	(SEQ ID	cagcca	NO: 5)		gcacaatccctc	20_R
				ID NO: 3)		(SEQ ID		NO: 7)	(SEQ ID			cctc (SEQ ID
						NO: 1)			NO: 2)			NO: 8)

The 5′ UTR is any UTR known in the art. For example, the 5′ UTR is polyAx30, polyAx120, PPT19, PPT19x4, GAAAx7, or polyAx30-EMCV. Preferably, the 5′ UTR is PPT19 or EMCV. Any known 3′ UTR may be used in the present invention; examples include HbB1-PolyAx10, HbB1, HbB1x2, or an Elastin-derived 3′ UTR (e.g., a motif from the Elastin 3′ UTR). Preferably, the 3′ UTR is an Elastin-derived 3′ UTR. Multiple tandem copies (e.g., 2, 3, 4, or more) of a UTR may be included in a nucleic acid (e.g., more than one copy of a motif from the Elastin 3′ UTR and more than one copy of the PPT19 5′ UTR). As used herein, the number after an “x” in a UTR's name refers to the number of copies of the UTR (or motif thereof). As an example, an Elastin 3′ UTR (or a motif thereof) that is repeated twice is referred to as Elastinx2 and an Elastin 3′ UTR (or a motif thereof) that is repeated three times is referred to as Elastinx3.
The 5′ and 3′ motifs identified by the inventors allow any size target RNA to be circularized. The RNA sequence is at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more nucleotides in length.
The RNA (e.g., mRNA) sequence may encode any protein of interest, for example the target RNA encodes for a hormone, scFv, single-domain antibody (also known as a nanobody), cytokine, intracellular protein, extracellular protein, tumor-associated antigen, chimeric antigen receptor, bacterial antigen, viral antigen, transposase, nuclease, or transcription factor. The RNA may encode a therapeutic polypeptide, e.g., preproinsulin, hypocretin, human growth hormone, leptin, oxytocin, vasopressin, factor VII, factor VIII, factor IX, erythropoietin, G-CSF, alpha-galactosidase A, iduronidase, N-acetylgalactosamine-4-sulfatase, FSH, DNase, tissue plasminogen activator, glucocerebrosidase, interferon, and IGF-1. The translated protein would have endogenous post-translational modifications and could be retained intracellularly or secreted. The RNA sequence may encode a polypeptide that comprises an epitope for presentation by an antigen presenting cell. The polypeptide may lead to improved (e.g., more efficient and greater quantity) T-cell priming, as determined by increased production of IFN-γ, including by proliferating cells.
The RNA sequence may be an RNA that is a reverse complement of an endogenous RNA, i.e., an mRNA, a miRNA, a tRNA, an rRNA, or a lncRNA; by “endogenous” is meant an RNA that is naturally transcribed by a cell. An RNA sequence that is a reverse complement may be referred to as a “non-coding RNA” since it does not encode a polypeptide. When an RNA sequence of the present invention binds an endogenous RNA, the endogenous RNA's function may be blocked or reduced; for example, when the endogenous RNA is an miRNA, the RNA sequence of the present invention prevents the miRNA from binding to its target mRNAs..
The RNA sequence may be capable of binding to an RNA-binding protein (RBP). When the RNA sequence binds an RBP, the nucleic acid of the present invention prevents the RBP from binding to its canonical linear RNA binding partner. Non-limiting examples of RBPs are found at the World Wide Web (www) at rbpdb.ccbr.utoronto.ca.
A circularized nucleic acid will have greater stability (i.e., more resistant to degradation or enzymatic digestion) than a nucleic acid having a similar sequence (e.g., identical or non-identical) but is non-circularized. The circularized nucleic acid will have greater stability in solution. A circularized nucleic acid will have greater stability in a cell, whether in vitro or in vivo (i.e., in an animal). By “greater stability” is meant a stability increase of 0.01%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, or more or any percentage therebetween. For example, a greater (as defined above) fraction of the starting amount of circularized nucleic acid will remain in a solution or a cell after a certain amount of time when under identical conditions (e.g., temperature and presence/absence of digestive enzymes) than a corresponding non-circularized nucleic acid.
A circularized nucleic acid may provide greater polypeptide translation (e.g., more polypeptide product and more efficient synthesis) relative to a nucleic acid having a similar sequence (e.g., identical or non-identical) but is non-circularized. By “greater polypeptide translation” is meant an increase of 0.01%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, or more or any percentage therebetween in the amount of polypeptide produced. For example, a greater (as defined above) number of polypeptides will be synthesized from a molecule of circularized nucleic acid than from a corresponding non-circularized nucleic acid.
A nucleic acid may comprise an internal ribosome entry site (IRES). Exemplary IRES sequences are listed at the World Wide Web at iresite.org. Preferably, the IRES is an encephalomyocarditis virus (EMCV) IRES or a PPT19 IRES.
A nucleic acid of the present invention may be in a cell (e.g., in vitro or in vitro in a non-human mammal). Non-limiting examples of cells include T cells, B cells, Natural Killer cells (NK), Natural Killer T (NKT) cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells.
A circularized nucleic acid of the present invention may be included in a composition, e.g., a pharmaceutical composition suitable for administration to a subject, e.g., a mammal, including a human. The composition may include both a circularized nucleic acid of the present invention and a nucleic acid having a similar sequence (e.g., identical or non-identical) but is non-circularized.

Methods for Circularizing RNA

The nucleic acid comprising the RNA sequence to be circularized can be produced by methods known in the art.
For example, primers can be designed to generate PCR templates suitable for in vitro transcription (IVT), for example by T7, T3, or S6 RNA polymerase. Preferably, the primers are designed with the following motifs:
Forward primer: {RNA polymerase promoter sequence-5′-(random nucleotides)-(5′CRC sequence)-(desired 5′ UTR)-(1^st20 nucleotides of desired RNA CDS)-3′}
Reverse primer: {5′-(random nucleotides)-(3′CRC sequence)-(reverse complement of desired 3′ UTR)-(reverse complement of last 20 nucleotides of desired RNA CDS)-3′}
Circularized RNA is produced by transcription of the PCR products generated with the above primers, or another set of primers, to produce RNA. The synthesized RNA is then treated to produce a 5′ monophosphate RNA. For example, 5′ monophosphate RNA is produced by treating the RNA with RNA 5′ pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase.
The 5′ monophosphate RNA is then enzymatically circularized for example with an RNA ligase such as T4 RNA ligase.
A nucleic acid of the present invention, which is non-circularized, may be circularized by ligating its 5′ terminus to its 3′ terminus. Ligating may be enzymatic, e.g., by a ligase. Preferably, the ligase is T4 RNA ligase.
Prior to ligation, a non-circularized nucleic acid is contacted with a phosphatase, e.g., RNA 5′ pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase, to produce a 5′ monophosphate RNA. Alternately, a non-circularized nucleic acid is contacted with a phosphatase, e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, and Calf Intestinal Phosphatase, and then contacted with a kinase, e.g., Polynucleotide Kinase.
A nucleic acid may undergo multiple (e.g., two, three, four, five, or more) rounds of ligation, thereby ensuring that the majority of nucleic acids, in a sample, is circularized, e.g., about 100%, about 90%, about 80%, about 70%, about 60%, about 51%, or any amount therebeweeen.
Optionally, non-circularized (i.e., linear) RNA is removed using an exonuclease to digest the linear RNA, e.g., RNase R, Exonuclease T, λ Exonuclease, Exonuclease I, Exonuclease VII, T7 Exonuclease, or XRN-1. Preferably, the exonuclease is RNase R and/or XRN-1.

Methods of Using Circularized RNA

The circularized RNA produced according to the methods of the invention are useful in gene therapy. In particular, the circularized RNA is useful for protein replacement therapy or in the production of RNA-based vaccines for an array of antigens. For example, the circularized RNA (e.g., mRNA) can encode tumor-associated antigens useful as cancer vaccines. In another aspect, the circularized RNA (e.g., mRNA) can encode a bacterial or viral antigen to prevent or alleviate a symptom of a bacterial or viral infection, e.g., as a vaccine. Additional embodiments include use of circularized RNA for use in cancer immunotherapies, infectious disease vaccines, genome engineering, genetic reprogramming, and protein-replacement/supplementation therapies.
Alternatively, the circularized RNA (e.g., mRNA) can encode a chimeric antigen receptor and be used to create a chimeric antigen receptor T-cell useful in immunotherapy. Chimeric antigen receptors (CARs) comprise binding domains derived from natural ligands or antibodies specific for cell-surface antigens, genetically fused to effector molecules such as the TCR alpha and beta chains, or components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. A CAR typically has an intracellular signaling domain, a transmembrane domain, and an extracellular domain.
The transmembrane and/or intracellular domain may include signaling domains from CD8, CD4, CD28, 4-1BB, OX40, ICOS, and/or CD3-zeta. The transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.
The transmembrane domain may further include a stalk region positioned between the extracellular domain (e.g., extracellular ligand-binding domain) and the transmembrane domain. The term “stalk region” used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain. A stalk region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence. In a preferred embodiment said stalk region is a part of human CD8 alpha chain.
The signal transducing domain or intracellular signaling domain of the CAR of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “signal transducing domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function. Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAM used in the invention can include as non limiting examples those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transducing domain of the CAR can comprise the CD3 zeta signaling domain, or the intracytoplasmic domain of the Fc epsilon RI beta or gamma chains.
The CAR may further include one or more additional costimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain, to further augment potency. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like. In some embodiments the intracellular signaling domain contains 2, 3, 4, or more costimulatory molecules in tandem.
The extracellular domain may include an antibody such as a Fab, a scFV, or a single-domain antibody (sdAb also known as a nanobody) and/or may include another polypeptide described herein. In a preferred embodiment, said extracellular ligand-binding domain is a single chain antibody fragment (scFv) comprising the light (VL) and the heavy (VH) variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker. Other binding domain than scFv can also be used for predefined targeting of lymphocytes, such as camelid single-domain antibody fragments (which are examples of an sdAb) or receptor ligands, antibody binding domains, antibody hypervariable loops or CDRs as non limiting examples.
As non limiting examples, the antigen of the CAR can be a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelia, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface markers.
A circularized nucleic acid of the present invention may encode a CAR and may be transfected or infected into a T-cell using any technique known in the art. A T-cell that expresses the CAR is referred to as a chimeric T-cell receptor cell (CART). The CART will express and bear on the cell surface membrane the chimeric antigen receptor encoded by the RNA sequence of a circularized nucleic acid of the present invention.
The present invention includes a nucleic acid encoding a CAR, methods for preparing a nucleic acid encoding a CAR, compositions comprising a nucleic acid encoding a CAR, methods for producing a CART, methods for treating a diseases using a CART, an isolated CART, and non-human mammals comprising a CART.
Any of the herein-described aspects or embodiments can be combined with any other aspect or embodiment described herein.

Definitions

The term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.
Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2′-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety as defined herein. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2′-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.
Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, and qucuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties, include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7 -deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide is also meant to include what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine. The term “nucleotide” is also meant to include the N3′ to P5′ phosphoramidate, resulting from the substitution of a ribosyl 3′ oxygen with an amine group. Preferably, the modified base is 5-methylcytidine (5mC).
Further, the term nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
The term “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T, respectively).
The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.
As used herein, the terms “mRNA” and “RNA” may be synonyms.
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides. “Isolated” or “purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises a significant percent (e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100%) of the sample in which it resides. In certain embodiments, a substantially purified component comprises at least 50%, 80%-85%, or 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. Generally, a substance is purified when it exists in a sample in an amount, relative to other components of the sample, that is not found naturally.
The term “oligonucleotide”, as used herein, denotes a single-stranded multimer of nucleotides from about 2 to 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 4 to 50 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be RNA oligonucleotides) or deoxyribonucleotide monomers. Oligonucleotides may be 5 to 20, 11 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 nucleotides in length, for example.
The term “duplex” or “double-stranded” as used herein refers to nucleic acids formed by hybridization of two single strands of nucleic acids containing complementary sequences. In most cases, genomic DNA is double-stranded.
The term “complementary” as used herein refers to a nucleotide sequence that base-pairs by non-covalent bonds to a target nucleic acid of interest. In the canonical Watson-Crick base pairing, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). As such, A is complementary to T and G is complementary to C. In RNA, A is complementary to U and vice versa. Typically, “complementary” refers to a nucleotide sequence that is at least partially complementary. The term “complementary” may also encompass duplexes that are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions. In certain cases, a nucleotide sequence may be partially complementary to a target, in which not all nucleotide is complementary to every nucleotide in the target nucleic acid in all the corresponding positions.
As defined herein, “RNA ligase” means an enzyme or composition of enzyme that is capable of catalyzing the joining or ligating of an RNA acceptor oligonucleotide, which has an hydroxyl group on its 3′ end, to an RNA donor, which has a 5′ phosphate group on its 5′ end. The invention is not limited with respect to the RNA ligase, and any RNA ligase from any source can be used in an embodiment of the methods and kits of the present invention. For example, in some embodiments, the RNA ligase is a polypeptide (gp63) encoded by bacteriophage T4 gene 63; this enzyme, which is commonly referred to simply as “T4 RNA ligase,” is more correctly now called “T4 RNA ligase 1” since Ho, C K and Shuman, S (Proc. Natl. Acad. Sci. USA 99: 12709-12714, 2002) described a second RNA ligase (gp24.1) that is encoded by bacteriophage T4 gene 24.1, which is now called “T4 RNA ligase 2.” Unless otherwise stated, when “T4 RNA ligase” is used in the present specification, is meant “T4 RNA ligase 1”. For example, in some other embodiments, the RNA ligase is a polypeptide derived from or encoded by an RNA ligase gene from bacteriophage TS2126, which infects Thermus scotoductus, as disclosed in U.S. Pat. No. 7,303,901 (i.e., bacteriophage TS2126 RNA ligase).
Linear nucleic acid molecules are said to have a “5′-terminus” (5′ end) and a “3′-terminus” (3′ end) because nucleic acid phosphodiester linkages occur at the 5′ carbon and 3′ carbon of the sugar moieties of the substituent mononucleotides. The end of a polynucleotide at which a new linkage would be to a 5′ carbon is its 5′ terminal nucleotide. The end of a polynucleotide at which a new linkage would be to a 3′ carbon is its 3′ terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3′- or 5′-terminus
“Transcription” means the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. The invention is not limited with respect to the RNA polymerase that is used for transcription. For example, a T7-type RNA polymerase can be used.
“Translation” means the formation of a polypeptide molecule by a ribosome based upon an RNA template.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes combinations of two or more cells, or entire cultures of cells; reference to “a polynucleotide” includes, as a practical matter, many copies of that polynucleotide. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless defined herein and below in the reminder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
Unless specifically stated or obvious from context, as used herein, the term “about”, is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
A “subject” in the context of the present invention is preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
As used herein, the term “encode” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.
For example, in some aspects, the term “encode” describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term “encode” also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.

EXAMPLES

Example 1: Circularized RNA Synthesis

RNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, #E2040S) according to manufacturer's instructions. 500-1000 ng of PCR product encoding the desired RNA sequence was used as template in these in vitro transcription (IVT) reactions. Synthesized RNA was then treated with RNA 5′ Pyrophosphohydrolase, or RppH, (NEB, #M0356S) to provide the 5′ monophosphate end necessary for enzymatic circularization. RppH-treated RNA was enzymatically circularized in reactions containing final concentrations of: 10% DMSO, 200 uM ATP, lx NEB Buffer 4, 40 U RNaseOUT (Life Technologies, #10777-019), and 30 U of T4 RNA Ligase 1 (NEB, #M0204L) for 2 hours at 37 ° C. Any remaining linear RNA in the circularization reactions was removed using the exonuclease Xrn-1 (NEB, #M0338L). After each step, reactions were purified using the GeneJet RNA Purification Kit (Thermo Scientific, #1(4082). Circularization was confirmed by running 500 ng of RNA product on a 6% polyacrylamide gel in 7 M Urea-TBE (Life Technologies, #EC6865) for 3 hours at 180 V, 4° C. Circularized product characteristically migrates slower than linear RNA, so a slower migrating band indicated circularized product when run alongside control non-circularized RNA. Additional confirmation was carried out using outward-oriented PCR (OOPCR), where primers are oriented outward from one other with respect to the linear template (as opposed to traditional PCR in which primers are oriented towards each other). cDNA was synthesized (Life Technologies, #4402954) from RNA samples and used as template in the OOPCR reactions. cDNA derived from non-circularized, linear RNA was used a negative control. An amplicon is generated solely from the circularized construct, as the polymerase can extend through the ligated ends.

Example 2: Circularized Generating CRC, 5′ And 3′ UTR Constructs

CRC sequences and experimental 5′/3′ UTRs were appended to RNA coding sequence (CDS) by generating PCR templates for IVT that had been amplified with primers of the following design:
Forward primer: 5′-(TAATACGACTCACTATAGGG)-(ttatgataac)-(tggctgcacgaattgcacaa)-(desired 5′ UTR)-(varied based on RNA CDS)-3′ (primer comprises SEQ ID NOS: 3, 4, and 2, respectively).
{5′-(RNA polymerase promoter sequence)-(random nucleotides)-(5′CRC sequence)-(desired 5′ UTR)-(1^st20 nucleotides of desired RNA CDS)-3′}.
Reverse primer: 5′-(agcgacttcg)-(ttgtgcaattcgtgcagcca)-(desired 3′ UTR)-(varied based on RNA CDS)-3′ (primer comprises SEQ ID NOs. 5 and 2, respectively).
{5′-(random nucleotides)-(3′CRC sequence)-(reverse complement of desired 3′ UTR)-(reverse complement of last 20 nucleotides of desired RNA CDS)-3′}
PCR templates generated with the above primers were used to generate circularized product as described in the below “Improvements to Existing Methodology for RNA Circularization” Example.

Example 3: Circularized Degradation Protection Assay

Degradation Protection Assay was carried out using 750 ng of RNA encoding nanoluciferase in 3 forms: linear RNA without a 5′ cap or 3′ PolyA tail (negative control), linear RNA with 5′ cap and 3′ PolyA tail (canonical mRNA), and circularized RNA. Each sample was incubated in water or 1% FBS for 0, 0.5, 1, 2, 4, or 8 hours at room temperature. Afterwards all samples were run on a 1% agarose gel for 35 minutes at 100 V.

Example 4: Circularized Measuring Translation Efficiency and RNA Stability

RNA constructs encoding nanoluciferase complexed with Lipofectamine® 2000 (Life Technologies, #11668) and transfected into Hep3B cells (human hepatocyte cell line) seeded at 10,000 cells/well in a 96-well plate. Protein expression kinetics were measured using the Nano-Glo Luciferase Assay System (Promega, #N1110) using samples taken at 24, 48, and 72 hours post-transfection.
To measure RNA stability, qPCR was carried out using samples derived from cells that had been transfected as described above. cDNA was synthesized at each time point using the Power SYBR® Green Cells-to-Ct Kit (Life Technologies, #4402954) according to the manufacturer's instruction. The housekeeping gene (3-actin was used to normalize the results.

Example 5: Improvements to Existing Methodology for RNA Circularization

Enzymatic circularization produces the highest levels of circularized product using existing protocols.
400, 500, and 600 nucleotide (NT) RNA were circularized using 3 previously established methods: enzymatic (Perreault, 1995), splint-mediated (Moore and Sharp, 1992), and self-splicing (Puttaraju, 1992). Circularized products were run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V (FIG. 1). Non-circularized RNA was run as a control. No circularized product was detected using the self-splicing method (data not shown). The data shows that use of a ligase, preferably T4 RNA ligase, efficiently generates circularized RNA.
RppH treatment yields the highest level of circularized product compared to other monophosphate-generating enzymes.
Circularization protocol was carried out using a 500 nucleotide (NT)-long template with the only variation being the enzyme(s) used to generate the monophosphate 5′ end necessary for T4 RNA Ligase-mediated circularization to occur. RNA 5′ Pyrophosphohydrolase (RppH) and apyrase carry out this reaction in a single step. Alternatively, a two-step reaction can be carried out in which a phosphatase is first used to removal all 3 phosphates on the 5′ end of the RNA molecule, followed by a second reaction in which a kinase adds a single phosphate back to the molecule. Final circularized products were denatured and then run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V (FIG. 2A). Approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 2B). The monophosphate enzyme that corresponded to the highest level of circularized product was chosen as the most efficient monophosphate enzyme. AP=Antarctic Phosphatase, rSAP=recombinant Shrimp Alkaline Phosphatase, CIP=Calf Intestinal Phosphatase, PNK=Polynucleotide Kinase. The data shows that RppH efficiently generates monophosphate RNA.
RppH is the most efficient monophosphate-generating enzyme.
500 nucleotide (NT) RNA (FIG. 3A) or 1000 NT RNA (FIG. 3B) treated with the RppH or Apyrase monophosphate-generating enzyme were circularized and then exposed to XRN-1, an exonulcease that digests only linear RNA with a monophosphate 5′ end. XRN-1 cannot degrade RNA with a triphosphate 5′ end (post-in vitro transcription linear RNA). Final products were denatured and then run on a TBE-Urea 5% polyacrylamide gel for 3 hours (FIG. 3A) or 4 hours (FIG. 3B) at 180 V. Remnant bands present in the apyrase-treated constructs indicate that this enzyme reaction does not go to completion. RppH produces much fainter or a complete lack of bands indicating that almost, if not all, molecules are modified by this enzyme. The data show that, among the tested enzymes, RppH is the most efficient enzyme for generating monophosphate RNA.
Performing multiple rounds of ligase reaction increases the yield of circularized product.
500 NT-long, circularized RNA was generated following the above-described protocol. After the initial circularization reaction was completed, circularized RNA was purified (Thermo, Genej et RNA Cleanup Kit) and used as template for two additional circularization reactions. After each round, 1 μg of RNA was set aside for further analysis. 1 μg of RNA from each round of circularization ( rounds 1, 2, and 3, respectively, Cl, C2, and C3) was run on a 2% E-gel (Invitrogen) along with non-circularized linear (L) control (FIG. 4A). The approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 4B). The data show that performing multiple rounds of ligase reaction increase the yield of circularized products.
RNase R and XRN-1 exonucleases result in the highest purity of circularized product.
5 μg of linear or circularized 500 NT-long RNA constructs were incubated for 1 hour with one of the indicated exonucleases. After exonuclease digestion, samples were denatured and run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V. RNase R and XRN-1 resulted in noticeable removal of linear RNA, although neither resulted in complete removal of residual linear RNA. The data show that use of RNase R and/or XRN-1 efficiently degrades linear RNA and thereby enriches for circularized RNA.

Example 6: Inclusion of CRC Sequences Enhances Efficiency of RNA Circularization.

Inclusion of CRC sequences enhances efficiency of circularization of RNA constructs 300 NT to 500 NT in length.
RNA molecules 300 NT, 400 NT, or 500 NT in length were generated with (+) or without (−) a 20-NT complement-reverse complement (CRC) sequence flanking the 5′ and 3′ ends of the molecule. Predicted secondary structures were generated using RNAFold software (FIG. 6A). Blue arrows point to the predicted orientation of the 5′ and 3′ ends of the RNA molecule. White boxes=no CRC; tan boxes=with CRC. All constructs underwent circularization reactions, were denatured, and were then run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V (FIG. 6B). The approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 6C). The data show that inclusion of CRC sequences enhances efficiency of RNA circularization.
RNA up to 1000 NT in length can be circularized when CRC sequences are included in the RNA molecule.
RNA molecules 600 NT, 700 NT, 800 NT, 900 NT, or 1000 NT in length were generated with (+) or without (−) a 20-NT complement-reverse complement (CRC) sequence flanking the 5′ and 3′ ends of the molecule. Predicted secondary structures were generated using RNAFold software (FIG. 7A). Blue arrows point to the predicted orientation of the 5′ and 3′ ends of the RNA molecule. White boxes=no CRC; tan boxes=with CRC. All constructs underwent circularization reactions, were denatured, and were then run on a TBE-Urea 5% polyacrylamide gel for 4 hours at 180 V (FIG. 7B). Circularized RNA products migrate slower on the gel (red arrows). Green arrows point to non-circularized, linear product. The approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 7C). Graphical representation of circularized product from PAGE gels illustrated in FIGS. 6 and 7 are shown in FIG. 7D. The data show that inclusion of CRC sequences enhances efficiency of RNA circularization for long RNA molecules.
Inclusion of CRC sequences enhances efficiency of circularization of RNA constructs up to at least 3000 NT in length.
Circularized RNA constructs of the indicated sizes (FIG. 8, Column A) were generated and subsequently digested with XRN-1. Since linear and circularized bands from RNA over 1000 NT cannot be adequately separated on denaturing polyacrylamide gel, the amount of circularized product was determined by first digesting the RNA with XRN-1. RNA concentrations pre- and post-XRN-1 digestion were determined via NanoDrop spectrophotometer. [(Remaining RNA post-XRN1)/(total starting RNA)×100] was used to calculate circularization efficiency. As a control, linear forms of each RNA construct, all containing triphosphate ends, were treated with XRN-1 to confirm the specificity of this enzyme, since XRN-1 cannot degrade RNA comprising a triphosphate 5′ end (Column B). To determine the efficiency with which XRN-1 removes linear RNA, monophosphate-treated linear forms of each RNA molecule were treated with XRN-1 (Column C). The average leftover product was ˜25%. This number was taken into account when determining the final circularization efficiency of each construct tested by first subtracting 25% from the remaining, post-XRN-1 RNA (Column D). The data show that inclusion of CRC sequences to enhance efficiency of RNA circularization and, in particular, for long RNA molecules.
Circularization efficiency is dependent upon the 5′ and 3′ end-positions of RNA molecules.
Circularized or linear RNA encoding the nanoluciferase reporter protein, flanked by a polyAx30 motif, HCV IRES, or PolyAx30-HCV IRES (5′ UTR) and a twice-repeated end sequence of the β-globin gene (3′ UTR) were generating with (+) or without (−) a 20-NT complement-reverse complement (CRC) sequence flanking the 5′ and 3′ ends of the molecule. Predicted secondary structures were generated using RNAFold software (FIG. 9A). Blue arrows point to the predicted orientation of the 5′ and 3′ ends of the RNA molecule. Tan boxes=with CRC; white boxes=no CRC. Constructs were denatured and run on a TBE-Urea 5% polyacrylamide gel for 4 hours at 180 V (FIG. 9B). CRC+ RNA constructs in either linear or circularized form were digested with RNase R, an exonuclease that targets free 3′ ends of linear RNA (FIG. 9C). L=Linear. C=Circular. Red arrows point to bands that represent circularized RNA. The presence of circularized product without a CRC sequence in the PolyA-containing RNA molecules and lack of circularized product in the HCV only RNA molecules evidences the importance of end position on circularization efficiency. This point is emphasized by the presence of circularized product in HCV-only RNA molecules when a CRC sequence is added and the increase in total circularized RNA when a CRC sequence is added to a PolyA-containing RNA molecule. Again, the data show that inclusion of CRC sequences enhances efficiency of RNA circularization, particularly among RNA molecules whose termini are not readily accessible to ligase.
Inclusion of CRC sequences results in robust and rapidly-produced levels of circularized product.
500 NT-long RNA molecules derived from the NLuc coding sequence (CDS) was incubated in circularization reactions for 0, 2, 4, 6, 8, 16, or 24 hours. Products were purified, denatured, and run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V (FIG. 10A). X=linear product not exposed to T4 RNA ligase. Red arrows point to bands that represent circularized RNA; green arrows point to bands that represent non-circularized, linear RNA. Approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 10B). The same circularization incubation times were carried out using a 1000 NT-long RNA molecule template. These constructs were run for 5 hours on the 5% polyacrylamide gel in order to see distinguishable separation of circularized and linear RNA (FIG. 10C). This extended run time resulted in warping of the RNA that made quantifying the percentage of circularized RNA impossible. Red arrows point to bands that represent circularized RNA; green arrows point to bands that represent non-circularized, linear RNA. Again, the data show that inclusion of CRC sequences enhances efficiency of RNA circularization
Circularized RNA constructs containing a longer CRC and shorter Random NT motif have the highest levels of circularization efficiency.
A panel of circularized RNA constructs encoding nanoluciferase that differed only in the length and composition of their CRC and random NT motifs were generated to test the effect that these motifs have on circularization efficiency. Diagram depicting the orientation of the CRC and random NT motifs and the predicted secondary structure these motifs confer when included in an RNA sequence (FIG. 11A). In total, 31 circularized RNA constructs with varying CRC and random NT motifs were generated, denatured, and run on a TBE-Urea 5% polyacrylamide gel for 3 hours at 180 V (FIG. 11B). Approximate percentage of circularized product for each construct was determined by quantifying band intensities using Image J software analysis (FIG. 11C). OG=original CRC/Random NT motif used in all other experiments (20 NT-long CRC, 10 NT-long random NT). A=indicates that the random motif is comprised entirely of a polyA sequence instead of a heterogeneous sequence of nucleotides. The data show that longer CRC and shorter random nucleotide overhangs yield more circularized product.
Shorter CRC results in higher translation efficiency of circularized RNA.
A panel of circularized RNA constructs encoding nanoluciferase that differed only in the length and composition of their CRC and random NT motifs were generated to test the effect that these motifs have on translation efficiency. Hep3B cells plated at 10,000 cells per well in a 96-well plate were transfected with each construct. Constructs were complexed to the transfection reagent Lipofectamine® 2000 (0.3 μL/well). Luciferase activity was measured 24 hours post transfection (FIG. 12). OG=original CRC/Random NT motif used in all other experiments (20 NT-long CRC, 10 NT-long random NT). A=indicates that the random motif is comprised entirely of a polyA sequence instead of a heterogeneous sequence of nucleotides. The data show that a shorter CRC confers enhanced translation efficiency of resulting circularized RNA.
Longer CRC results in higher IFN-B response in Hep3Bs transfected with circularized RNA.
A panel of circularized RNA constructs encoding nanoluciferase that differed only in the length and composition of their CRC and random NT motifs were generated to test the effect that these motifs have their immunogenicity. Hep3B cells plated at 10,000 cells per well in a 96-well plate were transfected with each construct. Constructs were complexed to the transfection reagent Lipofectamine® 2000 (0.3 μL/well). IFN-β levels measured by qPCR (normalized to β-actin) 24 hours post transfection (FIG. 13). OG=original CRC/Random NT motif used in all other experiments (20 NT-long CRC, 10 NT-long random NT). A=indicates that the random motif is comprised entirely of a polyA sequence instead of a heterogeneous sequence of nucleotides. These data show that a shorter CRC lowers interferon response of resulting circularized RNA.

Example 7: Characterizing Translation Efficiency of Circularized RNA In Vitro.

A 20-mer derived from the elastin 3′ UTR motif enhances translation efficiency of linear and circularized RNA.
Hep3B cells plated at 10,000 cells per well in a 96-well plate were transfected with 200 ng of linear or circularized RNA encoding the nanoluciferase protein. The transfection was performed using the transfection reagent Lipofectamine® 2000 (0.3 μL/well). The nanoluciferase coding sequence (CDS) was flanked by variable experimental 5′ and 3′ untranslated regions (UTRs). Luminescence levels were measured 24 hours post-transfection to determine differences in translation efficiency (FIG. 14). These data show that inclusion of at least one repeat of a 20-mer derived from the 3′ UTR of elastin (“Elastin”) in the circularized RNA construct increases translation efficiency.
The PPT19 (5′ UTR) and Elastinx3 (3′ UTR) combination sustain protein expression of circularized RNA.
Hep3B cells plated at 10,000 cells per well in a 96-well plate were transfected with 200 ng of linear or circularized RNA encoding the nanoluciferase protein complexed with the transfection reagent Lipofectamine 2000 (0.3 μL/well). The nanoluciferase coding sequence (CDS) was flanked by variable, experimental 5′ and 3′ untranslated regions (UTRs). Luminescence levels were measured 24, 48, and 96 hours post-transfection to determine differences in translation efficiency (FIG. 15). These data show that inclusion of the PPT19 sequence in a circularized RNA construct helps sustain protein expression.
A 20-mer elastin 3′ UTR motif, but not the HCV IRES (5′ UTR) or HbB1 (3′ UTR), enhances translation efficiency of circularized RNA.
Circularized RNA containing the nanoluciferase (NLuc) coding sequences flanked by experimental 5′ and 3′ UTRs were generated using the T7 High Yield RNA Synthesis Kit (NEB). Constructs were transfected into Hep3B cells and luminescence levels were measured 24 hours post-transfection to determine differences in translation efficiency (FIG. 16). Again, these data show that inclusion of at least one repeat of a 20-mer derived from the 3′ UTR of elastin in the circularized RNA construct increases translation efficiency.
Circularized RNA composed of 50% 5-methyl cytidine (5mC) modified nucleotides confers high levels of translation.
Circularized RNA containing the nanoluciferase (NLuc) coding sequences flanked by experimental 5′ and 3′ UTRs were generated using the T7 High Yield RNA Synthesis Kit (NEB). These in vitro transcription reactions where generating with either: 100% pseudouridine, 50% 5mC, or 50% pseudouridine and 50% 5mC. Constructs were transfected into Hep3B cells and luminescence levels were measured 24 hours post-transfection to determine differences in translation efficiency (FIG. 17). These data show that inclusion of modified nucleotides, such as 50% 5-methyl cytidine, improves translation efficiency.
Modified nucleotide panel reveals that 50% 5mC composition provides the highest level of translation efficiency to circularized RNA.
Circularized RNA encoding the nanoluciferase (NLuc) coding sequences flanked by experimental 5′ and 3′ UTRs were generated using the T7 High Yield RNA Synthesis Kit (NEB). Constructs were generated with the indicated modified nucleotide compositions. Constructs were transfected into Hep3B cells and luminescence levels were measured 24, 48, and 72 hours post-transfection to determine differences in translation efficiency (FIG. 18). Again, these data show that inclusion of modified nucleotides, such as 50% 5-methyl cytidine, improves translation efficiency.
EMCV IRES confers the greatest translation efficiency in human cells to circularized RNA but not to capped/tailed linear RNA.
A panel of NLuc-encoded RNA varying in their 5′ UTR containing experimentally verified IRES (World Wide Web at iresite.org) were transfected into Hep3B cells, and luminescence levels were measured 24 hours post-transfection to determine differences in translation efficiency. Three forms of RNA were transfected into Hep3Bs: canonical mRNA (containing a 5′ cap and polyA tail), unstable RNA (no cap or tail), or circularized RNA (FIG. 19). These data show that inclusion of EMCV IRES in the circularized RNA construct increases translation efficiency.
Spiking with a competitive cap analog confirms that translation of circularized RNA is cap-independent.
1.12 pmoles of linear or circularized NLuc RNA containing varying 5′ UTR motifs (EMCV, Non-IRES, or No UTR) were used as templates in rabbit reticulocyte (“retic”) lysate in vitro translation reactions. Prior to RNA addition, retic reactions were incubated for 15 minutes with 0 mM or 2 mM (final concentration) of ARCA cap analog at 30 degrees. Samples were incubated for another 75 minutes after addition of RNA. Reactions were transferred to a 96-well plate and assayed for NLuc expression by bioluminescence (FIG. 20). These data show that translation of linear RNA is compromised much more significantly than translation of circularized RNA (e.g., more than two-fold as much for EMCV-containing construct) upon addition of cap analog that competes for translation apparatus. That any inhibition is seen for the circularized RNA samples, which are expected to be translated in a cap-independent manner, suggests that 2 mM of cap analog is saturating and may induce conformational changes in eIF4E (and potentially eIF4G) that recruit other factors that are important to translation (e.g., eIF4A, eIF4B, and eIF3), thereby compromising their ability to find circularized RNA that is translationally competent even in the absence of the cap. Confirmatory experiments involving lower concentrations of cap analog may be performed using methods described herein.
Circularized RNA containing the EMCV IRES is translated more efficiently than canonical (cap+tailed) mRNA in a cell-free system.
1.12 pmoles of linear or circularized NLuc RNA containing varying 5′ UTR motifs (EMCV, PPT19x4, or No UTR) were used as templates in retic lysate in vitro translation reactions. At each indicated time point (over a 24 hour time course), a 15 μL aliquot was taken from each sample and assayed for NLuc expression by bioluminescence (FIG. 21). These data show that circularized RNA containing the EMCV IRES is translated more efficiently than canonical mRNA (e.g., comprising a 5′ cap and polyA tail) in a cell-free in vitro lysate system. It is noteworthy that no protein is produced from circularized RNA if no UTR is incorporated.

Example 8: Characterizing Stability of Circularized RNA

Circularized RNA is more resistant to degradation than canonical linear RNA is.
Degradation Protection Assay was carried out using 750 ng of RNA encoding nanoluciferase in 3 forms: linear RNA without a 5′ cap or PolyA tail (negative control), linear RNA with 5′ cap and PolyA tail (canonical mRNA), and circularized RNA. Each sample was incubated in water or 1% FBS for 0, 0.5, 1, 2, 4, or 8 hours at room temperature. Afterwards, all samples were run on a 1% agarose gel for 35 minutes at 110 V (FIG. 22). These data show that, in solution, circularized RNA is more stable than linear RNA, including canonical mRNA.
Circularized RNA encoding the therapeutic protein preproinsulin is sustained longer in vitro than linear RNA is.
Preproinsulin RNA was generated in 3 forms: linear RNA without a 5′ cap or PolyA tail (−/−), linear canonical mRNA with 5′ cap and PolyA tail (+/+), and circularized RNA (Circ.). Each form of RNA was generated with one of the following 5′ UTRs: Endogenous preproinsulin UTR, PolyAx120, or Polyx30. All constructs contained a 3′ UTR encoding a thrice-repeated 20-mer derived from the Elastin 3′ UTR. Constructs were transfected into Hep3B cells and preproinsulin RNA levels were measured by qPCR 24, 48, and 72 hours post-transfection (FIG. 23). These data show that, in cells, circularized RNA encoding a therapeutic protein is more stable than linear RNA, including canonical mRNA.
Circularized RNA is more stable than linear RNA is in vitro.
Degradation Protection Assay was carried out using 750 ng of RNA encoding nanoluciferase in 3 forms: linear RNA without a 5′ cap or PolyA tail (negative control), linear RNA with 5′ cap and PolyA tail (canonical mRNA), and circularized RNA. Each sample was incubated in water for 0, 24, 48, or 72 hours at room temperature. Afterwards, all samples were run on a 1% agarose gel for 35 minutes at 110 V. The data is summarized in FIG. 24. These data show that, in vitro, circularized RNA is more stable than linear RNA, including canonical mRNA.
Unlike linear RNA, circularized RNA shows minimal loss up to at least 3 days in vivo, in mouse liver following intravenous administration.
20 μg of linear or circularized RNA was intravenously injected into Balb/c mice. Mice were sacrificed 24, 48, or 72 hours post injection. Livers were isolated, total RNA extracted from livers (PureLink® RNA Mini Kit, Invitrogen), cDNA synthesized (Superscript® IV, Invitrogen) and qPCR carried out to measure changes in RNA levels over the 3-day time course (FIG. 25; NLuc levels normalized to β-actin). For linear RNA there was an approximate 75% decrease in absolute RNA level from day 1 to day 2. In contrast, transfected circularized RNA constructs maintained a relatively stable absolute RNA level throughout the assay time points. Furthermore, data also indicate that Nluc relative RNA level of circularized constructs have approximately 4-fold greater expression than linear constructs after 1 and 2 days. On the third day, the circularized RNA has approximately 2-fold greater expression than linear RNA. These data show that, in vivo, circularized RNA is more stable than linear RNA, including canonical mRNA.

Example 9: Characterizing Translation Efficiency of Circularized RNA In Vivo

In vivo, protein expression derived from circularized RNA is sustained longer than from linear RNA.
20, 40 or 60 μg of linear or circularized RNA injected intravenously into Balb/c mice. Bioluminescence levels of live mice were measured at 4 and 8 hours post injection with a Xenogen Imager (FIG. 26). For this, in vivo assays used an in vivo imaging system (IVIS), in which nanoluciferase protein can be detected in live mice. One of the advantages of using the IVIS detection system is that it is not an endpoint analysis and thus, the same mouse can be monitored for nanoluciferase protein production over an extended period of time. For these assays, the mice were injected with furimazine, which reacts with Nanoluc luciferase, resulting in the emission of light. The data from these experiments indicate that ample amounts of Nanaoluc luciferase were detected in the abdominal space, rather than in the spleen, 4 hours post i.v. injection of the linear RNA constructs. In contrast, the circularized RNA construct expressed little Nanoluc luciferase at the same 4-hour time point. However, after 8 hours, the linear RNA treated mice appeared to have ceased Nanoluc luciferase expression; it also appeared that the linear RNA construct had degreaded by the 8-hour time point as no signal was detected by IVIS. In contrast, circularized RNA treated mice seemed to have increased Nanoluc luciferase expression at the 8-hour time point (FIG. 26). These data show that, in vivo, protein production from circularized RNA is prolonged relative to linear RNA.
In vivo, circularized RNA is resistant to serum nucleases.
20 μg of circularized or linear RNA was injected into mice by hydrodynamic injection (1 mL in 5 seconds), either naked or complexed to the TransIT® transfection reagent (other transfection reagents, e.g., Invivofect®, TurboFect®, or RNAimax® can be used). NLuc protein expression was measured by in vivo imaging system (IVIS; Xenogen Imager), and average radiance levels were measured. Data show relative protein expression compared to the level observed 4 hours post-administration of RNA complexed to TransIT® (FIG. 27). Again, these data show that, in vivo, protein production from circularized RNA is prolonged relative to linear RNA.
It was found that RNA and protein levels were highest when TransIT® reagent was used. However, it was also noted that TransIT® can be toxic to mice, as half of the mice administered the TransIT® reagent died shortly after injection in one experiment. Optimal volumes of TransIT® reagent (i.e., volumes of the reagent that would not kill the mice, but yet allow for high RNA expression and protein function) were determined through a series of titration experiments. Four volumes of TransIT® were used in the titration experiments, 25 μL, 50 μL, 75 μL, and 100 μL. As a control for the titration experiments, untransfected animals as well as RNAimax®-transfected mice were used. Twenty-four hours following treatment, the mice were sacrificed and their livers were isolated and analyzed by qPCR for RNA level and for protein level (data not shown). These titration experiments indicated that mice had adverse reactions to volumes of 75 μL of TransIT® reagent. Based on the data obtained from these experiments, 50 μL of TransIT® was chosen for use, as this volume allows for clearly detectable RNA expression and protein function following transfection (data not shown) in the absence of acute toxicity to the mice.

Example 10: Circularized RNA Application in Cancer Vaccine Model

Peptide presentation continues to increase beyond two days if circularized RNA is transfected into Dendritic cells (DCs).
FIG. 28 shows the steps and reagents used to generate data in FIG. 29, which shows kinetics of peptide presentation following transfection of DCs with circularized and/or linear RNA. These data show that addition of circularized RNA to linear RNA (each encoding antigen) for transfection of antigen presenting cells results in continued increase in antigen presentation through day three post-transfection, whereas the presentation begins to decrease in the linear RNA-only sample at this time point.
Addition of circularized RNA to linear RNA for transfection of antigen presenting cells improves T cell priming.
FIG. 30 shows the steps and reagents used for co-culturing of OT.I CD8 T cells with RNA-transfected DCs. FIG. 31 shows the flow cytometry gating strategy used after co-culture of OT.I CD8 T cells with RNA-transfected DCs. FIG. 32 shows that addition of circularized RNA to linear RNA improved T cell priming (function). Higher levels of IFN-γ are produced by antigen-specific CD8⁺TCRβ⁺T cells upon co-culture with DCs transfected by a combination of Circular+Linear RNA. FIG. 33 shows that addition of circularized RNA to linear RNA improved T cell priming (specifically among proliferating cells). Higher levels of IFN-γ are produced by proliferating antigen-specific CD8⁺TCRβ⁺T cells upon co-culture with DCs transfected with the combination of Circular+Linear RNA. Together, these data show that addition of circularized RNA to linear RNA (each encoding antigen) for transfection of antigen presenting cells improves T cell priming, as evidenced by increased effector cytokine production, relative to transfection with either RNA alone.
Without being bound by theory, in the context of the combination of linear and circularized RNA, the linear RNA may act as a prime (based on a strong burst of expression), while the circularized RNA may act as a pseudo-boost (sustaining the presentation of antigen), thereby leading to improved effector T cell function.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1.-98. (canceled)

99. A method for producing a chimeric antigen receptor (CAR) cell comprising contacting an immune cell with a circularized nucleic acid, wherein the circularized nucleic acid comprises:

an internal ribosome entry site (IRES);

an RNA sequence encoding a chimeric antigen receptor (CAR); and

a 3′ UTR sequence.

100. The method of claim 99, wherein the immune cell is contacted with the circularized nucleic acid in vivo.

101. The method of claim 99, wherein the immune cell is contacted with the circularized nucleic acid in vitro.

102. The method of producing of claim 99, wherein the immune cell is contacted with the circularized nucleic acid and a transfection reagent.

103. The method of claim 99, wherein the immune cell comprises a T cell, B cell, Natural Killer (NK) cell, Natural Killer T (NKT) cell, mast cell, eosinophils, basophils, macrophages, neutrophils, dendritic cell, progenitor cell or precursor cells.

104. The method of claim 99, wherein the circularized nucleic acid comprises at least one non-complementary nucleotide sequence comprising 5 and 25 nucleotides located at the 5′ end or the 3′ end or both the 5′ end and the 3′ end.

105. The method of claim 99, wherein the circularized nucleic acid comprises at least one modified nucleotide.

106. The method of claim 99, wherein the circularized nucleic acid is produced from a non-circular precursor nucleic acid, and wherein the nucleic acid is circularized through ligation of the 5′ terminus of the non-circular precursor nucleic acid to the 3′ terminus of the non-circular precursor nucleic acid.

107. The method of claim 99, wherein the circularized nucleic acid comprises:

a 5′ complement-reverse complement (CRC) sequence;

a IRES sequence;

an RNA sequence encoding a chimeric antigen receptor (CAR);

a 3′ UTR sequence; and

a 3′ CRC sequence.

108. A pharmaceutical composition comprising the circularized nucleic acid produced by the method of claim 99 and a transfection reagent.

109. A chimeric antigen receptor (CAR) cell comprising an immune cell contacting with at least one circularized nucleic acid, wherein the circularized nucleic acid comprises:

an IRES sequence;

an RNA sequence encoding a chimeric antigen receptor (CAR); and

a 3′ UTR sequence.

110. The CAR cell of claim 109, wherein the CAR comprises at least one binding domain specific for a protein expressed on the surface of a tumor cell.

111. The CAR cell of claim 100, wherein the CAR comprises CD 19, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelia, a major histocompatibility complex (MHC) molecule, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) of fibronectin, extra domain B (EDB) of fibronectin, the Al domain of tenascin-C (TnC A1), fibroblast associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen.

112. The CAR cell of claim 109, wherein the CAR comprises at least one of the following signaling domains:

intracellular cellular domain;

signal transducing domain;

transmembrane domain; or

extracellular domain.

113. The CAR cell of claim 109, wherein the CAR comprises a transmembrane domain comprising a region derived from a 4-1BB, OX40, ICOS, CD8, CD4, CD28, or an antibody constant region.

114. The CAR cell of claim 109, wherein the CAR comprises an intracellular signaling domain comprising at least one cytoplasmic signaling sequence, wherein the cytoplasmic signaling sequence initiates antigen-dependent primary activation or provides a secondary or costimulatory signal in an antigen dependent manner.

115. The CAR cell of claim 109, wherein the cytoplasmic signaling sequence comprises an immunoreceptor tyrosine-based activation motif (ITAM) derived from a CD3 zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b or CD66d.

116. The CAR cell of claim 109, wherein the circularized nucleic acid has greater stability relative to a non-circularized nucleic acid.

117. A pharmaceutical composition comprising the CAR cell of claim 109 and a transfection reagent.

118. A pharmaceutical composition comprising a transfection reagent and a circularized nucleic acid encoding a chimeric antigen receptor (CAR), wherein the circularized nucleic acid comprises an IRES sequence, an RNA sequence encoding the CAR, and a 3′ UTR sequence, wherein the circularized nucleic acid has greater stability in vivo relative to a non-circularized nucleic acid.

119. A method for producing a polypeptide comprising transfecting an immune cell with a circularized nucleic acid, wherein the circularized nucleic acid comprises:

an internal ribosome entry site (IRES);

an RNA sequence encoding a polypeptide; and

a 3′ UTR sequence.

120. The method of claim 119, wherein the polypeptide comprises a tumor-associated antigen, cell-surface antigen, a chimeric antigen receptor, a T-cell receptor (TCR), a secreted protein, a bacterial or viral antigen, a transposase, nuclease, a transcription factor, a hormone, intracellular protein, extracellular protein, single-domain antibody, bacterial antigen, viral antigen, transposase, nuclease, an scFv, a Fab, a single -domain antibody, or a therapeutic protein.

121. The method of claim 120, wherein the therapeutic protein comprises a preproinsulin, hypocretin, human growth hormone, leptin, oxytocin, vasopressin, factor VII, factor VII, factor VIII, factor IX, erythropoietin, G-CSF, alpha-galactosidase A, iduronidase, N-acetylgalactosamine-4-sulfatase, FSH, DNase, tissue plasminogen activator, glucocerebrosidase, interferon, or IGF-1.

122. The method of claim 120, wherein the therapeutic protein comprises endogenous post-translational modifications.

123. The method of claim 120, wherein the therapeutic protein is retained intracellularly or secreted.

124. The method of claim 119, wherein the polypeptide comprises a protein capable of expression in an immune cell or an antigen presenting cell.

125. The method of claim 119, wherein the polypeptide leads to improved T-cell priming.

126. The method of claim 119, wherein the circularized nucleic acid is translated from a primer RNA sequence comprising of an RNA that is a reverse complement of an endogenous RNA.

127. The method of claim 119, wherein the circularization nucleic acid provides a greater polypeptide translation than a non-circularized nucleic acid.

128. A pharmaceutical composition comprising the polypeptide of claim 119.